Three loci have already been described learn more by Radtke et al. in a contemporary

study but were amplified here with other primers [32] (Table 2). For the SAG7 locus, no amplification was observed with primers directly flanking the TR for 14% (26/189) of the strains. A second primer pair targeting larger consensual flanking regions was designed to confirm the absence of the locus. PCR was performed in a final volume of 25 μl containing 10 ng DNA, 1 × PCR Reaction Buffer, 2 mM MgCl2 (Applied Biosystems), 5% DMSO (dimethyl sulfoxide), 1 unit of Taq DNA polymerase (Applied Biosystems), 200 μM of each dNTP and 0.5 μM of each flanking primer (Eurogentec, Belgium). Amplification was performed in a 2720 Thermal Cycler (Applied Biosystems) under the following conditions: initial denaturation for 5 min at 94°C, followed by 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 50°C and elongation for 60 s at 72°C plus a final elongation step for 7 min at 72°C. We separated 10 μl of PCR product by electrophoresis in a 2% agarose gel (Eurogentec, Belgium), which was also loaded with a 100 bp DNA size ladder (New England BioLabs). Electrophoresis was performed in 20 cm-long

gels, in 1× TBE buffer (89 mM Tris-Borate, 2.5 mM EDTA) containing 1 μg/ml ethidium check details selleck screening library bromide run at 10 V/cm. In each run, at least one lane was loaded with PCR product from one of the reference strains, NEM316, A909 or 2603 V/R. The gels were photographed under ultraviolet illumination, with Vision-Capt® Software

(Vilber-Lourmat, Marne la Vallée, France). The number of repeats for each VNTR was deduced from amplicon size, by comparison with the reference strain, for which the number of repeats was known. The allele number corresponded to the number of repeats. For the SAG7 locus, the lack of a VNTR was revealed by the absence of amplification with the first click here primer pair and the amplification of a fragment of the expected size with the second primer pair, which targeted larger consensual flanking regions. In this case, an allele number of 0 was given. For the SAG21 locus, a 117 bp PCR product was obtained, demonstrating deletion of the inserted sequence and, thus, the absence of a VNTR. An allele number of 0 was also assigned in this case. The MLVA genotype of a strain was expressed as its allelic profile, corresponding to the number of repeats at each VNTR, listed in the order SAG2, SAG3, SAG4, SAG7, SAG21, SAG22.